Methods to diagnose a required regulation of trophoblast invasion

ABSTRACT

Methods are provided for the diagnosis and treatment of patients with increased risk of preeclampsia. The methods involve measuring levels of TGF-β 3 , receptors of cytokines of the TGβ family, or HIF-1α.

FIELD OF THE INVENTION

[0001] The invention relates to methods and compositions for diagnosingand treating conditions requiring regulation of trophoblast invasion.

BACKGROUND OF THE INVENTION

[0002] During placental development the establishment of fetal-maternalinteractions is critical for a successful human pregnancy (1).Abnormalities of placenta formation due to shallow trophoblast invasionhave been linked to preeclampsia and fetal growth restriction (2). Incontrast, uncontrolled trophoblast invasion and abnormal trophoblastgrowth are associated with hydatiform mole and choriocarcinoma In thecourse of placenta formation, chorionic villous cytotrophoblasts undergotwo morphologically distinct pathways of differentiation. The vastmajority of cytotrophoblasts in both floating and anchoring villi fuseto form the syncyuotrophoblast layer, which permits gas and nutrientexchange for the developing embryo. A small percentage ofcytotrophoblasts in anchoring villi break through the syncytium, atselected sites, and generate columns of non-polarized cells whichmigrate into the endometrium. These extravillous trophoblasts (EVT)invade deeply into the uterus reaching the first third of the myometriumat which point they invade the spiral arteries, replacing theirendothelium and vascular wall. Invasion peaks at 12 weeks of gestationand rapidly declines thereafter, indicating that, unlike tumourinvasion, it is spatially and temporally regulated (3). Trophoblastinvasion in the decidua is accompanied by a complex modulation of thesynthesis and degradation of extracellular matrix (ECM) proteins and inthe expression of adhesion molecules (4-6). Along the invasive pathway,ECM proteins undergo changes in their spatial distribution with loss oflaminin and appearance of fibronectin (3, 4). EVT loose the expressionof E-cadherins. responsible for cell-cell adhesion between polarizedstem cytotrophoblasts, down-regulate α₆ β₄ integrin, a laminin receptor,and acquire α₅β₁ integrin, a fibronectin receptor (7). Once the EVTinvade the endometrium they express the α₁β₁ integrin, acollagen/laminin receptor. Thus, specific changes in ECM proteins andtheir receptors are associated with the acquisition of an invasivephenotype by the extravillous trophoblasts (4).

[0003] Preeclampsia occurs in 5-10% of pregnancies and is the leadingcause of death and illness in women during pregnancy. Preeclampsia isalso associated with considerable fetal/neonatal complications becauseof adverse intrauterine conditions and preterm delivery. There iscurrently no effective pharmacologic treatment for preeclampsia and theonly remedy is to remove the placenta (and hence deliver the fetuspreterm). Current protocols, including bedrest and antihypertensivedrugs, seek to stabilize maternal/fetal condition until delivery isnecessitated. It is estimated that around 200,000 children are bornpreterm in North America due to preeclampsia. Many of these babies willrequire costly intensive care at birth and if they survive may face alifetime of chronic illness (e.g. lung disease) or disability (e.g.cerebral palsy, mental handicaps, blindness). These conditions representa significant impact on subsequent requirements for community healthcare resources. Therefore, reducing the incidence of preeclampsia andpretern birth would have a tremendous positive impact on health caredelivery.

SUMMARY OF THE INVENTION

[0004] The present inventors have studied the mechanisms that regulatetrophoblast invasion. The inventors have found that antisense disruptionof the expression of the TGFβ receptor, endoglin, triggers invasion ofcytotrophoblast from first trimester villous explants in vitroindicating that the TGFβ receptor system, and in particular endoglin,plays a critical role in regulating this process. Significantly, thepresent inventors defined components that endogenously regulatetrophoblast invasion. TGF-β₃ was found to be a major regulator oftrophoblast invasion in vitro. In particular, the presence of TGF-β₃ andits receptors at 5-8 weeks at a time when there is no spontaneoustrophoblast invasion and the absence of these molecules at 12-13 weekswhen spontaneous invasion occurs, establishes a major role for TGF-β₃ asan endogenous inhibitor of trophoblast invasion. Down-regulation ofTGF-β₃ (but not β₁ or β₂) expression using antisense oligonucleotides,stimulated extravillous trophoblast cell (EVT) outgrowth/migration andfibronecun production in 5-8 villous explants indicating that TGF-β₃acts to suppress in vivo trophoblast invasion. The effects of anusensetreatment to TGF-β₃ are specific as they are prevented by addition ofexogenous TGF-β₁ but not TGF-β₁ or TGF-β₂. The stimulatory effects ofTGF-β₃ are lost after 9 weeks of gestation which is compatible withTGF-β₃ being produced by the villi during a specific window of gestationwithin the first trimester (5-8 weeks) and that inhibition of itssynthesis stimulates trophoblast differentiation. Addition of exogenousTGF-β₃ to the villous explants inhibits fibronectin synthesis.

[0005] The clinical importance of TGF-β₃ in regulating trophoblastinvasion has been highlighted by the finding that TGF-β₃ is highlyexpressed in trophoblast tissue of preeclamptic patients when comparedto that in age-matched control placenta while there was no change in theexpression of either the β₁ or β₂ isoform. Fibronectin and α₅ integrinexpression were also greater in preeclamptic placenta, indicating thatin preeclampsia, where there is shallow trophoblast invasion,trophoblast cells are arrested as an α₅ integrin phenotype producingTGF-β₃. These data are supported by the finding that villous explantsfrom a control (non-preeclamptic placenta, 32 weeks of gestation)spontaneously formed columns of trophoblasts that invaded thesurrounding Matrigel, while explants from a preeclamptic placenta didnot.

[0006] In contrast to TGF-β₃, acuvin, a TGF-β receptor, has been foundto trigger trophoblast invasion. Follistatin an activin binding protein,inhibited the stimulatory effect of activin, and antibodies andantisense to endoglin.

[0007] Oxygen tension was also found to play a role in regulatingtrophoblast invasion. The expression of the hypoxia inducible factor,HIF-1α, parallels that of TGF-β₃ in first trimester trophoblast (i.e.peaks at 6-8 weeks but decreases after 9-10 weeks when oxygen tensionincreases). Expression of HIF-1α was dramatically increased in placentasof preeclamptic patients when compared to age-matched control tissue.Induction of HIF-1α by low PO₂ (around 6-8 weeks) up regulates TGF-β₃transcription and blocks trophoblast invasion. A failure of the systemto down-regulate at 9-11 weeks (either due to a block in response tonormoxia or the absence of an increase in oxygen tension) leads toshallow invasion and predisposes to preeclampsia.

[0008] In addition to endoglin, the present inventors have found thatTGF-β₃ signals through a receptor complex which includes RI (ALK1), RIIand endoglin. While TGF-β RI (ALK-5) and TGF-β R-II are expressedthroughout the villi and decidua at 9-10 weeks gestation, they werefound to be absent from the base of the proximal columns of theanchoring villi at the transition zone between the villous and theinvading EVT exactly at the site where endoglin is up-regulated. Thisdramatic change in TGF-β receptor expression indicates that EVT withinthe columns in situ are not subject to the inhibitory actions of TGFβ,but via R-I and R-II they come under the control of this ligand uponentering the decidua. In addition, antisense induced disruption of RI(ALK-1) and RII expression stimulated trophoblast outgrowth/migrationand fibronectin synthesis. In contrast, antisense to RI (ALK-5)inhibited fibronectin synthesis.

[0009] Broadly stated the present invention relates to a method fordetecting, preventing, and/or treating a condition requiring regulationof trophoblast invasion by modulating (a) TGFβ₃ (b) receptors ofcytokines of the TGFβ family, (c) HIF-1α, and/or (d) O₂ tension. Inaccordance with one aspect of the invention a method is provided fordiagnosing in a subject a condition requiring regulation of trophoblastinvasion comprising detecting TGFβ₃, receptors of cytokines of the TGFβfamily, or HIF-1α, in a sample from the subject. In an embodiment of thediagnostic method of the invention, a method is provided for diagnosingincreased risk of preeclampsia in a subject comprising detecting TGFβ₃or its receptors, or HIF-1α in a sample from the subject.

[0010] The invention also broadly contemplates a method for regulatingtrophoblast invasion comprising inhibiting or stimulating TGFβ₃,receptors of cytokines of the TGFβ family, HIF-1α, or O₂ tension. In anembodiment of the invention, a method is provided for increasingtrophoblast invasion in a subject comprising administering to thesubject an effective amount of an inhibitor of (a) TGFβ₃, (b) receptorsof cytokines of the TGFβ family, and/or (c) HIF-1α. In a preferredembodiment of the invention a method is provided for treating a womansuffering from, or who may be susceptible to preeclampsia comprisingadministering therapeutically effective dosages of an inhibitor of (a)TGFβ₃, (b) receptors of cytokines of the TGFβ family, and/or (c) HIF-1α.A therapeutically effective dosage is an amount of an inhibitor of (a),(b) and/or (c) effective to down regulate or inhibit TGFβ₃ in the woman.

[0011] In another embodiment of the invention, a method is providing forreducing trophoblast invasion in a subject comprising administering aneffective amount of (a) TGFβ₃; (b) receptors of cytokines of the TGFβfamily; (c) HIF-1α; and/or (d) a stimulator of (a), (b) or (c). In apreferred embodiment, a method is provided for monitoring or treatingchoriocarcinoma or hydatiform mole in a subject comprising administeringtherapeutically effective dosages of (a) TGFβ₃; (b) receptors ofcytokines of the TGFβ family, (c) HIF-1α; and/or (d) a stimulator of(a), (b) or (c). An amount is administered which is effective to upregulate or stimulate TGFβ₃ in the subject.

[0012] The invention also relates to a composition adapted forregulating trophoblast invasion comprising a substance which inhibits orstimulates TGFβ₃, receptors of cytokines of the TGFβ family, and/orHIF-1α, or regulates O₂ tension, in an amount effective to inhibit orstimulate trophoblast invasion, and an appropriate carrier, diluent, orexcipient. In an embodiment of the invention, a composition is providedfor treating a woman suffering from, or who may be susceptible topreeclampsia, comprising a therapeutically effective amount of aninhibitor of (a) TGFβ₃, (b) receptors of cytokines of the TGFβ family,and/or (c) HIF-1α, and a carrier, diluent, or excipient. In anotherembodiment of the invention, a composition is provided for monitoring ortreating choriocarcinoma or hydatiform mole in a subject comprising atherapeutically effective amount of (a) TGFβ₃; (b) receptors ofcytokines of the TGFβ family; (c) HIF-1α; and/or (d) a stimulator of(a), (b) or (c), and a carrier, diluent, or excipient.

[0013] The invention further relates to a method of selecting asubstance that regulates trophoblast invasion comprising assaying for asubstance that inhibits or stimulates TGFβ₃, receptors of a cytokine ofthe TGFβ family, or HIF-1α. The substances may be used in the methods ofthe invention to regulate trophoblast invasion.

[0014] The invention also relates to kits for carrying out the methodsof the invention.

[0015] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will now be described in relation to the drawingsin which:

[0017]FIG. 1 shows the amino acid and nucleic acid sequence of TGFβ₃;

[0018]FIG. 2 shows the amino acid and nucleic acid sequence of HIF-1α;

[0019]FIG. 3A are Southern blots showing expression of TGF-β isoforms inhuman placenta in the first trimester of gestation:

[0020]FIG. 3B are photographs of immunoperoxidase staining of TGFβ₃performed in placental sections at 5, 8 and 12 weeks of gestation;

[0021]FIG. 4A are photographs showing that addition of recombinant TGFβ₃to antisense TGFβ₃ abolishes the antisense stimulatory effect ontrophoblast budding and outgrowth;

[0022]FIG. 4B are blots showing the reversal effect on antisense TGFβ₃stimulatory effect by exogenous TGFβ₃ for fibronectin synthesis;

[0023]FIG. 4C is a graph showing the changes in fibronectin estimatedafter normalization to control cultures;

[0024]FIG. 4D are blots showing the effects on gelatinase activity inconditioned media of explants treated with sense or antisenseoligonucleotides to TGFβ₃;

[0025]FIG. 4E are blots showing that the antisense TGFβ₃ stimulatoryeffect on fibronectin production is lost after 9 weeks of gestation;

[0026]FIG. 5A are blots showing message expression of TGFβ isoforms, α₅integrin receptor, and fibronectin in preeclamptic and age-matchedcontrol placentae;

[0027]FIG. 5B are photographs of immunoperoxidase staining of TGFβ₃performed in placental sections from normal pregnancies and pregnanciescomplicated by preeclampsia;

[0028]FIG. 6A are photographs showing that antisense oligonucleotides toTGFβ₃ induces the formation of trophoblast cells in preeclamptic villousexplants;

[0029]FIG. 6B shows the results of gelatin Zymography of explants of 32weeks gestation from preeclamptic placentae treated with antisense orcontrol sense oligonucleotides to TGFβ₃ for 5 days;

[0030]FIG. 6C are Western blots with MMP9 antisera of explants of 32weeks gestation from preeclamptic placentae treated with antisense orcontrol sense oligonucleotides to TGFβ₃ for 5 days;

[0031]FIG. 7A is a blot showing expression of HIF-1α placenta in thefirst trimester of gestation;

[0032]FIG. 7B is a blot showing expression of HIF-1α in preeclamptic(PE) and age-matched control placenta (C);

[0033]FIG. 8 is a blot showing the effect of low oxygen tension on TGFβ₃and HIF-1α expression in villous explants;

[0034]FIG. 9 are photographs at 20% O₂ and 3% O₂ (25× and 50×) showingthe effect of low oxygen tension on villous explant morphology; and

[0035]FIG. 10 are photographs showing the effect of antisense to HIF-1αon villous explant morphology.

DETAILED DESCRIPTION OF THE INVENTION

[0036] 1. Diagnostic Methods

[0037] As hereinbefore mentioned, the present invention provides amethod for diagnosing in a subject a condition requiring regulation oftrophoblast invasion comprising detecting TGFβ₃, receptors of cytokinesof the TGFβ family, or HIF-1α in a sample from the subject. In anembodiment of the diagnostic method of the invention, a method isprovided for diagnosing increased risk of preeclampsia in a subjectcomprising detecting TGFβ₃, its receptors, or HIF-1α in a sample fromthe subject.

[0038] TGFβ₃ is a cytokine of the TGFβ family and it has the structuralcharacteristics of the members of the TGFβ family. TGFβ is produced as aprecursor characterised by having an N-terminal hydrophobic signalsequence for translocation across the endoplasmic reticulum, apro-region, and a C-terminal bioactive domain. Prior to release from thecell, the pro-region is cleaved at a site containing four basic aminoacids immediately preceding the bioacive domain (Massague, 1990,Annu.Review, Cell Biol. 6:597).

[0039] The precursor structure of TGFβ is shared by members of the TGFβfamily, with the exception of the TGFβ 4 precursor which lacks adistinguishable signal sequence. The degree of identity between familymembers in the C-terminal bioactive domain is from 25 to 90% (See Basleret al. Cell, 73:687, 1993, FIG. 2). All nine cysteines are conserved inthe bioactive domain in the TGFβ family. The bioactive domain is cleavedto generate a mature monomer.

[0040] The TGFβ family includes five members, termed TGFβ 1 through TGFβ5, all of which form homodimers of about 25 kd (reviewed in Massague,1990). The family also includes TGFβ 1.2 which is a heterodimercontaining a β1 and a β2 subunit linked by disulfide bonds. The fiveTGFβ genes are highly conserved. The mature TGFβ processed cytokinesproduced from the members of the gene family show almost 100% amino acididentity between species, and the five peptides as a group show about60-80% identity. The amino acid sequence and nucleic acid sequence ofTGFβ₃ are shown in FIG. 1 (See also sequences for GenBank Acession Nos.HSTGF31-HSTGF37, and HSTGFB3M).

[0041] “Receptors of cytokines of the TGFβ family” or “TGFβ receptors”refers to the specific cell surface receptors which bind to cytokines ofthe TGFβ family, in particular TGFβ₃, including the TGF-β type Ireceptor (ALK-1 or ALK-5) (R-I), TGF-β type II receptor (R-II),betaglycan, endoglin and activin, and complexes of the receptors, inparticular a RI-RII-endoglin complex. Endoglin binds TGFβ₁ and β₃ withhigh affinity (K_(D)=50 pM). Betaglycan has considerable sequencehomology to endoglin (Chiefetz, S., et al J. Biol Chem. 267: 19027,1992; Lopez-Casillas, F., et al. Cell 67:785, 1991; Wang, X. F., et al,Cell 67:797, 1991), it can bind all three forms of TGFβ₃, and itregulates access of the ligands to R-I and R-II which areserine/threonine kinases and unlike betaglycan, are necessary for signaltransduction (Wrana, J. L. et al, Cell 71:1003, 1992. Lopez-Casillas etal, Cell 73:1435, 1993; Franzen, P., et al Cell 75:681, 1993; Laiho, M.et al, J. Biol. Chem. 266:9108; Massague, J. et al, Trends Cell Biol.4:172, 1994). TGFβR-II is an integral membrane protein which contains ashort extrocellular domain, a single transmembrane domain, and anintracellular serine/threonine kinase domain (Lin H. Y. et al., Cell68:775, 1992). Serine/threonine kinases encoding type II receptors havebeen cloned which are structurally related to the type II receptors(Wrana, J. L. et al. Cell 71:1003, 1992, ten Dikje, P., et al, Oncogene8:2879, 1993; Ebner, R., et al Science 260:1344, 1993; Ebner, R., et alScience 262:900, 1993). TGFβ R-I (human ALK-5), binds TGFβ₁ and β₃ onlyin the presence of TGFβ R-II (Wrana, J. L. et al. Cell 71:1003, 1992).The human ALK-1 (TGFβ R-I) binds TGFβ when forming a heterodimericcomplex with TGFβ R-II (Franzen, P., et al Cell 75:681, 1993). TGFβ R-IIkinase, which is endogenously phosphorylated, phosphorylates andactivates R-I which then initiates further downstream signals (Wrana, J.L. et al, Nature 370:341, 1994).

[0042] Hypoxia-inducible factor-I (HIF-1) is present in nuclear extractsof many mammalian cells cultivated in a low oxygen atmosphere (SemenzaG. L. et al Mol. Cell. Biol. 12:5447, 1992; Wang, G. L. et al J. Biol.Chem. 268:21513, 1993). HIF-I binds as a phosphoprotein to a short DNAmotif (BACGTSSK) identified in the 3′-flanking regions of manyhypoxia-induced genes (Semenza, G. L. et al. J. Biol Chem 269:23757,1994; Liu, Y., et al Circulation Res. 77:638, 1995; Firth, J. D. et alProc. Natl. Acad. Sci. USA 91:6496, 1994; Abe, M. et al. Anal. Biochem.216:276, 1994). HIF-I binds DNA as a heterodimeric complex composed oftwo subunits of the inducible HIF-1α and the constitutively expressedHIF-Iβ.

[0043] TGFβ₃, receptors of cytokines of the TGFβ family (c.g.TGFβ RI(ALK-1), TGFβ RII, or a complex of RI-RII-endoglin), or HIF-1α may bedeteted in a variety of samples from a patient. Examples of suitablesamples include cells (e.g. fetal or maternal); and, fluids (fetal ormaternal), including for example, serum, plasma, amniotic fluid, saliva,and conditioned medium from fetal or maternal cells.

[0044] TGFβ₃, receptors of cytokines of the TGFβ family, or HIF-1α maybe detected using a substance which directly or indirectly interactswith the cytokine, TGFβ receptors, or HIF-1α. For example, antibodiesspecific for TGFβ₃, the TGFβ receptors, or HIF-1α may be used todiagnose and monitor a condition requiring regulation of trophoblastinvasion. A method of the invention using antibodies may utilizeCountercurrent Immuno-Electrophoresis (CIEP), Radioimmunoassays,Radioimmunoprecipitations, and Enzyme-Linked Immuno-Sorbent Assays(ELISA), Dot Blot assays, Inhibition or Competition assays and sandwichassays (see U.S. Pat. Nos. 4,376,110 and 4,486,530).

[0045] Antibodies used in the methods of the invention includemonoclonal antibodies, polyclonal antibodies, antibody fragments (e.g.,Fab, and F(ab′)₂ and recombinantly produced binding partners. Polyclonalantibodies may be readily generated by one of ordinary skill in the artfrom a variety of warm-blooded animals such as horses, cows, variousfowl, rabbits, mice, or rats. Monoclonal antibodies may also be readilygenerated using conventional techniques (see U.S. Pat. Nos. RE 32,011,4,902,614, 4,543,439, and 4,411,993 which are incorporated herein byreference; see also Monoclonal Antibodies, Hybridomas: A New Dimensionin Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol(eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane(eds.). Cold Spring Harbor Laboratory Press, 1988, which are alsoincorporated herein by reference). Binding partners may be constructedutilizing recombinant DNA techniques to incorporate the variable regionsof a gene which encodes a specifically binding antibody (See Bird etal., Science 242:423-426, 1988).

[0046] Antibodies may also be obtained from commercial sources. Forexample, antibodies to TGFβ₃ may be obtained from American DiagnosticsInc., CT. USA, Oncogene Science, NY, USA, and Dimension Laboratones,Mississauga, Canada

[0047] The presence of TGFβ₃ in a sample may also be determined bymeasunng the binding of the cytokine to compounds which are known tointeract with TGFβ₃ such as its receptors, or decorin, thrombospondin,the serum glycoprotein α2-macroglobulin, fctuin, or thyroglobulin (Y.Yamaguchi, D. M. Mann, E. Ruoslahti, Nature 346, 281 (1990); S.Scholtz-Cherry J. E. Murphy-Ullrich, J. Cell Biol. 122, 923 (1993);O'Conner-McCourt, L. M. Wakefield J. Biol. Chem. 262, 14090 (1987); andJ. Massague Curr. Biol. 1, 117 (1991)). These compounds art referred toherein as “TGFβ Binding Compounds”.

[0048] The presence of receptors of cytokines of the TGF family may bedetermined by measuring the binding of the receptors to molecules (orparts thereof) which are known to interact with the receptors such astheir ligands. In parucular, peptides derived from sites on ligandswhich bind to the receptors may be used. A peptide derived from aspecific site on a ligand may encompass the amino acid sequence of anaturally occurring binding site, any portion of that binding site, orother molecular entity that functions to bind an associated molecule. Apeptide derived from such a site will interact directly or indirectlywith an associated receptor molecule in such a way as to mimic thenative binding site. Such peptides may include competitive inhibitors,enhancers, peptide mimetics, and the like as discussed below.

[0049] The presence of HIF-1α may be determined by measuring the bindingof HIF-α1 to DNA molecules which are known to interact with HIF-α1 suchas hypoxia inducing genes. The TGFβ binding compounds and molecules thatinteract with the receptors and HIF-1α are referred to herein as“Binding Compounds”.

[0050] The antibodies specific for the TGFβ₃, TGFβ receptors, or HIF-1α,or the Binding Compounds may be labelled using conventional methods withvarious enzymes, fluorescent materials, luminescent materials andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase: examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin: anexample of a luminescent material includes luminol; and examples ofsuitable radioactive materials include radioactive phosphorous ³²P,iodine I¹²⁵, I¹³¹ or tritium.

[0051] An antibody to TGFβ₃, a TGFβ family receptor, or HIF-1α, or aBinding Compound may also be indirectly labelled with a ligand bindingpartner. For example, the antibodies, or a TGFβ₃ Binding Compound may beconjugated to one partner of a ligand binding pair, and the TGFβ₃ may becoupled to the other partner of the ligand binding pair. Representativeexamples include avidin-biotin, and riboflavin-riboflavin bindingprotein. Preferably the antibodies are biotinylated. Methods forconjugating the antibodies discussed above with the ligand bindingpartner may be readily accomplished by one of ordinary skill in the art(see Wilchek and Bayer, “The Avidin-Biotin Complex in BioanalyticalApplications.” Anal. Biochem. 171:1-32, 1988).

[0052] The antibodies or Binding Compounds used in the method of theinvention may be insolubilized. For example, the antibodies or BindingCompounds may be bound to a suitable carrier. Examples of suitablecarriers are agarose, cellulose, dextran, Sephadex, Sepharose,carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin,plastic film, plastic tube, glass beads, polyamine-methyl,vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleicacid copolymer, nylon, silk, etc. The carrier may be in the shape of,for example, a tube, test plate, beads, disc, sphere etc. Theinsolubilized compound or antibodies may be prepared by reacting thematerial with a suitable insoluble carrier using known chemical orphysical methods, for example, cyanogen bromide coupling.

[0053] Indirect methods may also be employed in which a primaryantigen-antibody reaction is amplified by the introduction of a secondantibody, having specificity for the antibody reactive against thecytokine. By way of example, if the antibody having specificity againstTGFβ₃ is a rabbit IgG antibody, the second antibody may be goatanti-rabbit gamma-globulin labelled with a detectable substance asdescribed herein.

[0054] TGFβ₃, TGFβ receptors, or HIF-1α can also be assayed in a sampleusing nucleotide probes to detect nucleic acid molecules encoding aTGFβ₃, the TGFβ receptors, or HIF-1α. Suitable probes include nucleicacid molecules based on nucleic acid sequences encoding TGFβ₃, the TGFβreceptors, or HIF-1α. A nucleotide probe may be labelled with adetectable substance such as a radioactive label which provides for anadequate signal and has sufficient half-life such as ³²P, ³H, ¹⁴C or thelike. Other detectable substances which may be used include antigensthat are recognized by a specific labelled antibody, fluorescentcompounds, enzymes, antibodies specific for a labelled antigen, andluminescent compounds. An appropriate label may be selected havingregard to the rate of hybridization and binding of the probe to thenucleotide to be detected and the amount of nucleotide available forhybridization. Labelled probes may be hybridized to nucleic acids onsolid supports such as nitrocellulose filters or nylon membranes asgenerally described in Sambrook et al. 1989, Molecular Cloning, ALaboratory Manual (2nd ed.).

[0055] A nucleic acid molecule encoding TGFβ₃, TGFβ receptors, or HIF1αcan also be detected by selective amplification of the nucleic acidmolecules using polymerase chain reaction (PCR) methods. Syntheticoligonucleotide primers can be constructed from the sequences of theTGFβ₃, TGFβ receptors, or HIF1α using conventional methods. A nucleicacid can be amplified in a sample using these oligonucleotide primersand standard PCR amplification techniques.

[0056] In a preferred embodiment of the invention, a method is providedfor diagnosing increased risk of preeclampsia in a subject comprisingdetecting TGF-β₃, TGFβ R-I (ALK-1). TGFβ R-II, endoglin, HIF-1 α, or acomplex of R-I (ALK-1)-R-II-endoglin in a sample, and in particularusing antibodies specific for TGF-β₃. Levels of TGF-β₃, TGFβ receptorsor complexes thereof, or HIF-1α may be measured during the firsttrimester of pregnancy (approximately 1 to 14 weeks). It is preferredthat at least two measurements be taken during this period, preferablyincluding a measurement at about 14 to 16 weeks. If the levels aresignificantly increased as compared to levels typical for women who donot suffer from preeclampsia the patient is diagnosed as having anincreased risk of suffering preeclampsia. Levels above those typical forwomen who do not suffer from preeclampsia may be suspect and furthermonitoring and measurement of TGFβ₃, TGFβ receptors, or HIF-1α may beappropriate. The information from the diagnostic method may be used toidentify subjects who may benefit from a course of treatment, such astreatment via administration of inhibitors of TGFβ, as discussed herein.

[0057] It will also be appreciated that the above methods may also beuseful in the diagnosis or monitoring of chonocarcinoma or hydatiformmole which involves uncontrolled trophoblast invasion (i.e. may beassociated with abnormally low levels of TGFβ₃, TGFβ family receptors,or HIF1α). Further the above methods may be used to diagnose or monitorother pregnancy complications including intrauterine growth restriction,molar pregnancy, preterm labour, preterm birth, fetal anomalies, andplacental abruption. The diagnostic and monitoring methods of theinvention may also involve determining responsiveness of cells tooxygen.

[0058] The invention also relates to kits for carrying out the methodsof the invention. The kits comprise instructions, negative and positivecontrols, and means for direct or indirect measurement of TGFβ₃, TGFβreceptors, or HIF1α.

[0059] 2. Regulation of Trophoblast Invasion in a Subject

[0060] The invention also provides a method of regulating trophoblastinvasion comprising directly or indirectly inhibiting or stimulating (a)TGFβ₃, (b) receptors of cytokines of the TGFβ family, (c) HIF1α; and/or(d) O₂ tension, preferably inhibiting or stimulating TGF-β₃. Trophoblastinvasion may also be regulated by optimizing oxygenation of tissues.

[0061] In an embodiment of the invention, a method is provided forincreasing trophoblast invasion in a subject comprising administering aneffective amount of a substance which is an inhibitor of (a) TGF-β₃, (b)receptors of cytokines of the TGFβ family, and/or (c) HIF-1α. Inparticular, methods are provided for treating a women suffering from orwho may be susceptible to preeclampsia.

[0062] In another embodiment of the invention, a method is providing forreducing trophoblast invasion in a subject comprising administering aneffective amount of (a) TGFβ₃; (b) receptors of cytokines of the TGFβfamily; (c) HIF-α1; and/or (d) a stimulator of (a), (b) or (c). Themethod may be used to monitor or treat choriocarcinoma or hydatiformmole.

[0063] The methods of the invention may also be used to monitor or treatother complications of pregnancy such as intrauterine growthrestriction, molar pregnancy, preterm labour, preterm birth, fetalanomalies, or placental abruption.

[0064] Substances that regulate trophoblast invasion can be selected byassaying for a substance that inhibits or stimulates the activity ofTGF-β₃, TGFβ receptors, or HIF-1α. A substance that regulatestrophoblast invasion can also be identified based on its ability tospecifically interfere or stimulate the interaction of (a) TGF-β₃ and areceptor for the cytokine (e.g. the interaction of TGFβ₃ and endoglin,or TGF-β₃ and R-I, R-II, or a complex of R-I-R-II endoglin, or (b)TGF-β₃ and HIF-1α.

[0065] Therefore, a method is provided for evaluating a compound for itsability to regulate trophoblast invasion comprising the steps of:

[0066] (a) reacting TGFβ₃ or a part thereof that binds to a receptor ofa cytokine of the TGFβ family, with a receptor of a cytokine of the TGFβfamily or a part thereof that binds to TGFβ₃, and a test substance,wherein the TGFβ₃ and receptor of a cytokine of the TGFβ family or partsthereof, are selected so that they bind to form a ligand-receptorcomplex: and

[0067] (b) comparing to a control in the absence of the substance todetermine the effect of the substance.

[0068] In particular, a method is provided for identifying a substancewhich regulates trophoblast invasion comprising the steps of:

[0069] (a) reacting TGFβ₃ or a part thereof that binds to a receptor ofa cytokine of the TGFβ family, and a receptor of a cytokine of the TGFβfamily or a part thereof that binds to TGFβ₃, and a test substance,wherein the TGFβ₃ and receptor of a cytokine of the TGFβ family or partsthereof, are selected so that they bind to form a ligand-receptorcomplex, under conditions which permit the formation of ligand-receptorcomplexes, and

[0070] (b) assaying for complexes, for free substance, for non-complexedTGFβ₃ or receptor, or for activation of the receptor.

[0071] The substance may stimulate or inhibit the interacuon of TGFβ ora part thereof that binds the TGFβ receptor, and the TGFβ receptor.

[0072] In an embodiment of the invention a receptor complex is employedcomprising TGFβ R-I (ALK-1)-TGFβ RII-endoglin.

[0073] Activation of the receptor may be assayed by measuringphosphorylation of the receptor, or by assaying for a biological affecton a cell, such measuring biochemical markers of trophoblast invasionsuch as cell proliferation. FN synthesis, integrin expression, upregulation of gelatinase and type IV collagenase expression andactivity.

[0074] The invention also provides a method for evaluating a substancefor its ability to regulate trophoblast invasion comprising the stepsof:

[0075] (a) reacting TGFβ₃ or a part of TGFβ₃ that binds to HIF-1α,HIF-1α or a part of the protein that binds to TGFβ₃, and a testsubstance, wherein the TGFβ₃ or part thereof, and HIF-1α or part thereofbind to form a TGFβ3-HIF-1α complex; and

[0076] (b) comparing to a control in the absence of the substance todetermine the effect of the substance.

[0077] The substance may stimulate or inhibit the interaction of TGFβ₃and HIF-1α, or the activation of TGFβ by HIF-1α and thereby regulatetrophoblast invasion.

[0078] The substances identified using the methods of the inventioninclude but are not limited to peptides such as soluble peptidesincluding Ig-tailed fusion peptides, members of random peptide librariesand combinatorial chemistry-derived molecular libraries made of D-and/or L-configuration amino acids, phosphopeptides (including membersof random or partially degenerate, directed phosphopeptide libraries),antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic,chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab)₂, and Fabexpression library fragments, and cpitope-binding fragments thereof)],and small organic or inorganic molecules. The substance may be anendogenous physiological compound or it may be a natural or syntheticcompound. The substance may be a TGFβ R-I-TGFβ R-II-endoglin complex,which competitively inhibits the binding of TGFβ₃ to its naturalreceptors. The invention contemplates isolated TGFβ R-I-TGFβR-II-endoglin complexes and their use in regulating trophoblastinvasion.

[0079] The substances may be peptides derived from the binding sites ofTGF-β₃ and a receptor for the cytokine such as endoglin, R-I or R-II, ora complex of R-I-R-II-endoglin: or the binding sites of TGF-β₃ andHIF1α. A peptide derived from a specific binding site may encompass theamino acid sequence of a naturally occurring binding site, any portionof that binding site, or other molecular entity that functions to bindan associated molecule. A peptide derived from such a binding site willinteract directly or indirectly with an associated molecule in such away as to mimic the native binding domain. Such peptides may includecompetitive inhibitors, enhancers, peptide mimctics, and the like. Allof these peptides as well as molecules substantially homologous,complementary or otherwise functionally or structurally equivalent tothese peptides may be used for purposes of the present invention.

[0080] “Peptide mimetics” are structures which serve as substitutes forpeptides in interactions between molecules (See Morgan et al (1989),Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimeticsinclude synthetic structures which may or may not contain amino acidsand/or peptide bonds but retain the structural and functional featuresof a peptide, or enhancer or inhibitor of the invention. Peptidemimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc.Natl. Acad. Sci USA 89:9367); and peptide libraries containing peptidesof a designed length representing all possible sequences of amino acidscorresponding to a peptide of the invention.

[0081] Peptides may be synthesized by conventional techniques. Forexample, the peptides may be synthesized by chemical synthesis usingsolid phase peptide synthesis. These methods employ either solid orsolution phase synthesis methods (see for example. J. M. Stewart, and J.D. Young, Solid Phase Peptide Synthesis. 2^(nd) Ed., Pierce ChemicalCo., Rockford Ill. (1984) and G. Barany and R. B. Merrifield, ThePeptides: Analysis Synthesis. Biology editors E. Gross and J. MeienhoferVol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phasesynthesis techniques; and M Bodansky, Principles of Peptide Synthesis,Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., ThePeptides: Analysis, Synthesis, Biology, supra, Vol 1, for classicalsolution synthesis.)

[0082] Peptide mimetics may be designed based on information obtained bysystematic replacement of L-amino acids by D-amino acids, replacement ofside chains with groups having different electronic properties, and bysystematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptoidsmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

[0083] A substance that regulates trophoblast invasion may be a moleculewhich interferes with the transcription and/or translation of TGFβ₃,TGFβ receptors, or HIF-1α. For example, the sequence of a nucleic acidmolecule encoding TGFβ₃, TGFβ receptors (e.g. endoglin. R-I (ALK-1),R-II, or RI-RII-endoglin complex), or fragments thereof, may be invertedrelative to its normal presentation for transcription to produce anantisensc nucleic acid molecule. An anusense nucleic acid molecule maybe constructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. Examples of antisense molecules forTGFβ₃ are 5′-CCTTTGCAAGTGCATC-3′ and 5′-GATGCACTTGCAAAGG-3′.

[0084] The treatment methods and compositions described herein may usesubstances that are known inhibitors of TGFβ₃. For example, antibodiesto TGFβ₃, the TGFβ Binding Compounds including decorin,α2-macroglobulin, fetuin, and thyroglobulin, or peptides derived fromthe sites on these compounds that bind to TGFβ₃, or chimeras of thesemolecules may be employed.

[0085] Activin, another member of the TGFβ receptor family, triggerstrophoblast invasion and therefore it may be used to enhance trophoblastinvasion in a subject.

[0086] The utility of a selected inhibitor or stimulator may beconfirmed in experimental model systems. For example, the human villousexplant culture system described by Genbacev et al. (21) can be used toconfirm the utility of an inhibitor for treatment of preeclampsia.

[0087] In a preferred embodiment of the invention a method is providedfor treating a woman suffering from, or who may be suspectible topreeclampsia comprising administering therapeutically effective dosagesof an inhibitor of TGF-β₃ or TGFβ receptors, an inhibitor of HIF-1α, ora substance identified in accordance with the methods of the invention.Preferably treatment with the inhibitor begins early in the firsttrimester, at about 10 to about 16 weeks, and may continue untilmeasured TGF-β₃ levels, TGF-β receptor levels, or HIF-1α levels arewithin the normal range. Preferably, treatment with the inhibitor orsubstance is not continued beyond about 30 weeks of gestation. For thepurposes of the present invention normal TGF-β₃ levels, TGFβ receptorlevels, or HIF-1α levels are defined as those levels typical forpregnant women who do not suffer from preeclampsia. Treatment with theinhibitor is discontinued after TGF-β₃ levels, TGF-β receptor levels,and/or HIF-1α levels are within normal range, and before any adverseeffects of administration of the inhibitor are observed.

[0088] One or more inhibitors or one or more stimulators of TGFβ₃, TGFβreceptors, or HIF-1α, or substances selected in accordance with themethods of the invention including Binding Compounds, may beincorporated into a composition adapted for regulating trophoblastinvasion. In an embodiment of the invention, a composition is providedfor treating a woman suffering from, or who may be susceptible topreeclampsia, comprising a therapeutically effective amount of aninhibitor of TGFβ₃, TGFβ receptors, or HIF-1α, or substance selected inaccordance with the methods of the invention including TGFβ BindingCompounds, and a carrier, diluent, or excipient.

[0089] The compositions of the invention contain at least one inhibitoror stimulator of TGF-β₃, TGFβ receptors, or HIF-1α, or substanceidentified in accordance with the methods of the invention, alone ortogether with other active substances. Such compositions can be fororal, parenteral, or local use. They are therefore in solid or semisolidform, for example pills, tablets, and capsules.

[0090] The composition of the invention can be intended foradministration to subjects such as humans or animals. The pharmaceuticalcompositions can be prepared by per se known methods for the preparationof pharmaceutically acceptable compositions which can be administered topatients, and such that an effective quantity of the active substance iscombined in a mixture with a pharmaceutically acceptable vehicle,carrier or diluent. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton. Pa., USA 1985).

[0091] The compositions of the invention may be administered togetherwith or prior to administration of other biological factors that havebeen found to affect trophoblast proliferation. Examples of thesefactors include IL-11 (Ireland et al Blood 84:267a, 1994), G-CSF, GM-CSFand M-CSF (U.S. Pat. No. 5,580,554 to Keith).

[0092] The compositions and other biological factors may be administeredthrough any known means. Systemic administration, such as intravenous orsubcutaneous administration is preferred. A therapeutically effectiveamount of an active ingredient e.g. inhibitor is an amount effective toelicit the desired therapeutic response but insufficient to cause atoxic reaction. The dosage for the compositions is determined by theattending physician taking into account factors such as the condition,body weight, diet of the subject, and the time of administration.

[0093] For example, a therapeutically effective dose of an inhibitor,e.g. an amount sufficient to lower levels of TGFβ₃ to normal levels, isabout 1 to 200 μg/kg/day. The method of the invention may involve aseries of administrations of the composition. Such a series may takeplace over a period of 7 to about 21 days and one or more series may beadministered. The composition may be administered initially at the lowend of the dosage range and the dose will be increased incrementallyover a preselected time course.

[0094] An inhibitor or stimulator of TGFβ₃, receptors of cytokines ofthe TGFβ family, or HIF-1α, or substance identified in accordance withthe methods of the invention may be administered by gene therapytechniques using genetically modified trophoblasts or by directlyintroducing genes encoding the inhibitors or stimulators of TGFβ₃, orreceptors of cytokines of the TGFβ family, or substances intotrophoblasts in vivo. Trophoblasts may be transformed or transfectedwith a recombinant vector (e.g. retroviral vectors, adenoviral vectorsand DNA virus vectors). Genes encoding inhibitors or stimulators, orsubstances may be introduced into cells of a subject in vivo usingphysical techniques such as microinjection and electroporation orchemical methods such as coprecipitation and incorporation of DNA intoliposomes. Antisense molecules may also be introduced in vivo usingthese conventional methods.

[0095] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLE 1

[0096] Materials and Methods

[0097] Establishment of Human Trophoblast Villous Explant Culture

[0098] Villous explant cultures were established from first trimesterhuman placentae by a modification of the method of Genbacev et al. (21).First trimester human placentae (5-8 weeks gestation) were obtained fromelective terminations of pregnancies by dilatation and curettage.Placental tissue was placed in ice-cold phosphate buffered saline (PBS)and processed within two hours of collection. The tissue was washed insterile PBS, and aseptically dissected using a microscope to removeendometrial tissue and fetal membranes. Small fragments of placentalvilli (15-20 mg wet weight) were teased apart and placed on atransparent Biopore membrane of 12-mm diameter Millicell-CM culture dishinserts with a pore size of 0.4 μm (Millipore Corp. Bedfords Mass.). Theinserts were precoated with 0.2 ml of undiluted Matrigel (CollaborativeResearch Inc), polymenzed at 37° C. for 30 min. and transferred in a24-well culture dish. Explants were cultured in DMEM/F12 (Gibco, GrandIsland, N.Y.) supplemented with 100 μg/ml streptomycin, 100 U/mlpenicillin and 0.25 μg/ml ascorbic acid, pH 7.4. Culture media werechanged every 48 h and collected for measurement of human chorionicgonadotropin (hCG) and progesterone. Villous explants were kept inculture for up to 6 days. Flattening of the distal end of the villoustips, their adherence to Matrigel and the appearance of extravilloustrophoblast cells (EVT) breaking through from the tips, were used asmarkers of morphological integrity and trophoblast differentiation aspreviously described by Genbacev et al. (21). EVT cell outgrowth andmigration were consistently monitored and quantutated using the ratio ofEVT outgrowths/villous tip, where the nominator, EVT outgrowths,represents the number of extravillous trophoblast columns sprouting fromthe villous tips plus the number of islands of EVT invading into theMatrigel. The denominator represents the total number of villous tips ina single explant culture. EVT outgrowth from the distal end of thevillous tips and their migration into the surrounding matrix wereobserved for up to 6 days in culture.

[0099] Initial experiments, in the presence of 10% (v/v) fetal bovineserum (FBS), demonstrated that DMEM/F12 supported greater EVT sproutingand migration than DMEM. In order to study the effect of various agentson EVT differentiation, a serum-free villous explant culture system wasdeveloped. Villous explants of 5-8 weeks gestation were incubatedovernight in DMEM/F12 or DMEM/F12+10% (v/v) FBS to promote attachment ofthe distal villous tips to the Matrigel. Following this incubationperiod, explants were washed with fresh medium and cultured in eitherserum-free DMEM/F12 or DMEM/F12 supplemented with varying concentrationsof FBS (0.5% and 10%). In serum-free medium EVT/villous tip was1.58±0.08 while it was 1.32±0.17 in 0.5% FBS and 1.26±0.02 in 10% FBS(mean±s.e.m. of 3 separate experiments, each carried out in triplicate),suggesting that villous explant cultures were viable for at least 6 daysin a serum-free medium. All subsequent experiments were performed withDMEM/F12 in the absence of serum.

[0100] The viability of the explant cultures was assessed by measuringhCG and progesterone production rate in the culture media collected atthe time of media change every 48 h. Both hCG and progesteroneconcentrations were measured by Radioimmunoassays (Coat-A-Count HCG IRMAand progesterone; DPC, Los Angeles, Calif.). Results are expressed forprogesterone as ng/0.1 g wet weight tissue and for hCG as IU/0.1 g wetweight tissue.

[0101] Antibodies

[0102] Murine monoclonal antibody (MAb) 44G4 specific for human endoglinwas produced as previously described (22). IgG purified from ascites wasused in all functional assays. Rat MAb 7D3 against cytokeratin was agift from Drs. S. Fisher and C. Damsky (San Francisco, Calif., USA).Murine MAb TS2/7 against the α₁ integrin subunit was provided by Dr. M.Hemler (Boston, Mass. USA). Mouse MAb P1D6 against the α₅ integrinsubunit was from Chemicon (Temecula, Calif.); rat MAb GoH3 against theα₆ integrin subunit was purchased from Serotec Canada (Toronto, Ont.Canada) and the neutralizing rabbit polyclonal antibody to TGF-β wasfrom R&D (Minneapolis, Minn.). Purified mouse IgG from Coulter (Hialeah,Fla.) and rat IgG from Sigma (Diagnostic, Toronto, Ont. Canada) wereused as negative controls.

[0103] Immunohistochemistry

[0104] Villous explants kept in culture for 6 days in the presence orabsence of antisense oligonucleotides to endoglin were dissected awayfrom the insert membrane with the supporting Matrigel. Explants andplacental tissue of 9 weeks gestation were fixed for 1 h at 4° C. in 4%(vol/vol) paraformaldehyde, cryoprotected by incubation in 10% (vol/vol)glycerol for 30 min and 50% (vol/vol) OCT compound (Tissue Tek. Miles,Ind.) for 18 h, embedded in 100% OCT and frozen in liquid nitrogen. Tenmicron sections were cut with a cryostat and mounted on poly-L-lysinecoated slides. To verify the quality of the tissue and select the mostrepresentative sections, every tenth one was stained with haematoxylinand cosin; neighbouring sections were selected and stained using theavidin-biotin immunoperoxidase method. Endogenous peroxidasce enzymeactivity was quenched with 3% (vol/vol) hydrogen peroxide in 0.01 MTris-HCl, pH 7.4, containing 0.15 M NaCl, or methanol for 10 minutes.Non-specific binding sites were blocked using 5% (vol/vol) normal horseserum (NHS) and 1% (wt/vol) BSA in Tris-buffer for 40 min at 23° C. Inthe case of murine monoclonal antibodies, a higher background wasobserved and it was necessary to preincubate the sections with 5%(wt/vol) texas red-conjugated goat anti-mouse IgG antibody for 1 h at23° C. prior to incubation with primary antibody at 4° C. for 1 h.Optimal antibody concentrations were established in preliminaryexperiments by titration and were used as follows: 44G4, 5 μg/ml; rabbitanti-TGF-β, 20 μg/ml; P1D6, 20 μg/ml; GoH3, 0.5 μg/ml; TS2/7, 20 μg/ml;7D3, 10 μg/ml. The slides were washed three times with Tris-buffer, thenincubated with a 200-fold dilution of biotinylated goat anti-rabbit IgGor a 300-fold dilution of biotinylated horse anti-mouse or anti-rat IgG,for 1 h at 4° C. After washing three times with Tris-buffer, the slideswere incubated with an avidin-biotin complex for 1 h. Slides were washedagain in Tris-buffer and developed in 0.075% (wt/vol)3,3-diaminobenzidine in Tris-buffer, pH 7.6, containing 0.002% (vol/vol)H₂O₂ giving rise to a brown product. After light counterstaining withtoluidine blue, slides were dehydrated in an ascending ethanol series,cleared in xylene, and mounted. In control experiments, primaryantibodies were replaced with non-immune mouse or rat IgG, or blockingsolution [5% (vol/vol) NGS and 1% (wt/vol) BSA].

[0105] Effect of Antibody to Endoglin on EVT Formation

[0106] Villous explants, prepared from placentae of 5-8 weeks gestationwere incubated for 16 h in DMEM/F12. Explant cultures were then washedwith fresh serum-free medium and incubated in serum-free DMEM/F12 mediumcontaining increasing concentrations of MAb 44G4 IgG (0.1 to 10 μg/ml).DMEM/F12 medium±antibody was replaced every 48 h. Antibody addition wasthus performed on day 1, 3 and 5 of culture. Morphological integrity ofvillous explants and their EVT differentiation were monitored daily forup to 6 days.

[0107] Antisense Oligonucleotides and Their Effects on EVT Formation

[0108] Phosphorothioate oligonucleotides (ON) were synthesized on a DNAsynthesizer and purified by capillary electrophoresis. Oligonuclcotidesof 16 base pairs targeted against sequences adjacent to the AUGinitiation codon of human endoglin (23) mRNA were synthesized. Previousstudies have demonstrated that antisense oligonucleotides, targeted tosequences adjacent to initiation codons, are most efficient ininhibiting translation (24). Furthermore, 16-mer oligonucleotides areshort enough to be taken up efficiently and provide sufficientspecificity for hybridization to the corresponding target mRNA (24). Thesequences of the antisense and sense endoglin oligonucleotidcs were5′-GCGTGCCGCGGTCCAT-3′ and 5′-ATGGACCGCGGCACGC-3′, respectively. Anoligomer with the same composition as the antisense oligonucleotide, butwith a scrambled sequence, 5′-GCGGGCCTCGTTCCAG-3′, was also synthesizedand used as a negative control. Oligonucleotides were dissolved in waterand their concentration was estimated by optical density at OD₂₆₀.Antisense or sense oligonucleotides (5-10 μM) were added to the villousexplants on day 1 and day 3 of culture. EVT sprouting and migration fromthe distal end of the villous tips were recorded daily for up to 6 days.

[0109] Fibronectin Production

[0110] Villous explants of 5-8 weeks gestation were incubated overnightin DMEM/F12. Explants were then washed and incubated in DMEM/F12containing either 10 μg/ml MAb 44G4 or non-immune IgG, 10 μM antisense,scrambled or sense endoglin oligonucleotides. The medium with or withoutthe various agents was changed on day 3 and was replaced on day 5 bymethionine-cysteine free low glucose DMEM containing 25 μCi/ml of[³⁵S]methionine/cysteine with or without the same antibodies oroligonucleotides. The cultures were metabolically labelled for 18 h.Conditioned culture media were collected and diluted with an equalamount of 25 mM Tris-HCl buffer, pH 7.4, 0.15 M NaCl and 0.5% (v/v)Triton X-100 and fibronectin was isolated using gelatin-Sepharose aspreviously described (25). Briefly, 50 μl of the gelatin-Sepharosesuspension was added to 500 μl of medium and the samples were incubatedovernight at 4° C. The gelatin-Sepharose beads were centrifuged, washedthree times in Tris/Triton X-100 buffer and fibronectin was eluted byboiling for 5 min in 1% (v/v) SDS and electrophoresed on a 4-12% (w/v)polyacrylamide gradient gels. Radiolabeled fibronectin was revealed byautoradiography and quantitated using a PhosphoImager (410A and ImageQuant software, Molecular Dynamics).

[0111] [³H]Thymidine Incorporation into DNA

[0112] Villous explants of 5-8 weeks gestation, cultured for 48 h withand without antisense ON to endoglin, were incubated in the presence of1 μCi of [³H]thymidine per milliliter of medium. After 6 h of incubationexplants were washed with PBS, fixed in 4% paraformaldehyde for 1 h.embedded in OCT and processed for cryostat sections as previouslydescribed. Ten micron sections were mounted on3-amino-propyl-tryethoxysilane-precoated slides and coated with NBT-2emulsion (Eastman Kodak, Rochester, N.Y.). Slides were developed after 3days using Kodak D-19 developer, counterstained with cosin and examinedby bright-field microscopy.

[0113] Data Analysis

[0114] All data are presented as means±s.e.m. of at least three separateexperiments carried out in triplicate. Statistical significance wasdetermined by Student's (t)-(test for paired groups and by one-wayanalysis of variance followed by assessment of differences usingStudent-Newman-Keuls test for non-paired groups. Significance wasdefined as p<0.05.

[0115] Results

[0116] Stimulation of EVT Outgrowth and Migration by Antibody andAntisense Oligonucleotides to Endoglin

[0117] The morphological examination of villous explants of 5-8 weeksgestation, cultured in serum-free medium, revealed a pattern of EVTdifferentiation (cell outgrowth and migration) similar to that describedby Genbacev et al (21). The viability of the explants, as measured bythe rate of production of progesterone and hCG, remained relativelyconstant for up to 6 days.

[0118] The ability of an antibody to endoglin (MAb 44G4) to alter theearly events of EVT differentiation along the invasive pathway wasexamined. Exposure of villous explants of 5-8 weeks gestation to 44G4IgG was associated with an increase in EVT outgrowth from the distal endof the villous tips and a higher number of cells migrating into thesurrounding matrix. Stimulation of EVT outgrowth and migration by 44G4IgG was specific as incubation of explants with an equivalent amount ofnon-immune murne IgG or medium alone had no effect. Furthermore additionof 44D7 IgG (10 μg/ml) reactive with CD98 antigen expressed at highlevels on syncytrophoblast (26) had no stimulatory effect.

[0119] Antisense endoglin also enhanced the number of EVT outgrowths aswell as their migration and invasion into the Matrigel. Controlexplants, cultured in the presence of sense endoglin oligonucleotides,exhibited no such effect.

[0120] Further experiments demonstrated that 24 h after the addition of44G4 IgG (day 2 of culture) there was a significant increase in EVToutgrowth and migration from 0.20±0.03 in the control group to 2.03±0.46in the antibody treated group (n=4; p<0.005). After 5 days of treatment(day 6) the number of EVT outgrowths increased from 0.64±0.09 in controlIgG-treated explants to 3.2±0.5 in the 44G4 IgG-treated explants (n=10,p<0.05). Subsequent experiments demonstrated that the stimulatory effectof 44G4 IgG was dose-dependent and maximal at 1 μg/ml.

[0121] The stimulatory effect of antisense endoglin oligonucleotides onEVT outgrowth and migration was observed on day 3 of culture with6.87±1.5 in the antisense-treated group versus 1.42±0.41 in thesense-treated group (p<0.05). After 5 days of exposure, the number ofEVT/villous tip increased from 2.08±0.47 in sense-treated explants to8.46±1.7 in antisense-treated cultures. The antisense-endoglin effect ontrophoblast differentiation was specific as incubation of explants withan equivalent amount of either sense endoglin or scrambledantisense-endoglin oligonucleotide (not shown) had no effect. Antisenseendoglin stimulated EVT outgrowth and migration in aconcentration-dependent manner with maximal stimulation observed at 10μM.

[0122] Characterization of Trophoblast Differentiation Along theInvasive Pathway in Villous Explants Culture

[0123] Previous reports indicate that stem trophoblasts within thevillous core and at the proximal site of the column, where trophoblastsstart to migrate away from the stem villi, undergo proliferation (21),whereas differentiated EVT do not. Therefore, studies were carried outto determine if EVT outgrowth triggered by antisense endoglin treatmentwas due to cell division or migration. [³H]Thymidine autoradiography ofexplants exposed to antisense endoglin ON showed villous trophoblastproliferation within the villous tip at the proximal site of the formingcolumn, while both differentiated EVT, which have invaded thesurrounding Matrigel, and mesenchymal cells in the villous core did notshow any DNA synthesis. This suggests that EVT within the column do notdivide and that blockage of endoglin most likely induces cell migrationfrom the villous core.

[0124] Trophoblast differentiation in situ is accompanied by atemporally and spatially regulated switch in integrin repertoire (4).When placental explants of 5-8 weeks gestation were maintained inculture for 5 days in the presence of antisense-endoglinoligonucleotides, the stimulation of EVT outgrowth and migration wasalso accompanied by changes in integrin expression. The α₆ integrinsubunit was found on polarized cytotrophoblasts within the villi and onthe non-polarized trophoblasts in the proximal columns. The α₅ integrinsubunit was minimally expressed on polarized trophoblasts or syncytium,but was present on EVT within the columns. EVT which had migratedfurther away in the Matrigel were negative for the α₅ integrin. Alltrophoblast cells, including CTB within the villi, thesyncytiotrophoblast and EVT stained positively for cytokeraun confirmingthe epithelial-like nature of the cells forming the columns andmigrating into the Matrigel. EVT which have migrated into Matrigel werepositive for the α₁ integrin. A polyclonal antibody to TGF-β showedstaining of the syncytiotrophoblast and stroma of the villi, suggestingthat TGF-β was present in the culture system. Migrating EVT and theMatrigel itself, known to contain TGF-β, showed weak positive staining.No reactivity was observed in the explants stained with control IgG.

[0125] As little EVT outgrowth is observed under basal cultureconditions, the expression of endoglin in trophoblast columns could onlybe studied in antisense-treated explants. Immunohistochemical analysisof explants treated with antisense oligonucleotides to endoglin revealedthat in intact villi the syncytiophoblast maintained high levels ofendoglin. Low levels of endoglin and α₅ integrin were observed in thestroma; however this staining appears non-specific as it was alsoobserved with non-immune IgG. The staining of endoglin in EVT ofexplants treated with antisense endoglin was weakly positive whencompared to sections of the same explant stained with control IgG. Inaddition, endoglin expression in proximal columns of explants was muchreduced when compared to sections of 9 weeks gestation placenta stainedunder similar conditions. When a subsequent section of this placenta isstained for α₅ integrin, the transition zone in the proximal column isclearly visualized as negative for α₅, but positive for endoglin. The α₅integrin in explants treated with antisense endoglin was also found tobe highly expressed in EVT within proximal and distal columns. Thesedata suggest that antisense endoglin treatment, which promotes EVToutgrowth and migration in explant cultures, induces a decrease inendoglin expression at the level of the transition zone, which isfollowed by an increase in the expression of the α₅ integrin fibronectinreceptor.

[0126] Stimulation of Fibronectin Production by Interference with TGF-βResponse

[0127] FN has been localized to specific regions of the matrixsurrounding the anchoring villi and its production is increased duringEVT differentiation (27). Thus the effect of either 44G4 IgG orantisense endoglin on fibronectin synthesis by villous explants from 5-8weeks gestation was investigated. Explants were metabolically labelledon day 4 with [³⁵S]methionine and newly synthesized FN released into themedia over a period of 18 h was measured. Both 44G4 IgG andantisense-endoglin oligonucleotides induced a significantly greaterproduction of FN than that observed in control IgG or senseoligonucleotide-treated cultures. Phospholmager analysis of all datademonstrated a 8- and 5-fold increase in FN synthesis (5 independentexperiments carried out in triplicate, p<0.05) for 44G4 IgG andantisense-endoglin treated explants, respectively, relative to controlsense or DMEM/F12 alone. FN production in villous explants, cultured inthe presence of a scrambled antisense endoglin oligonucleotide, wassimilar to that observed in sense-treated explants or in medium alone.

[0128] To demonstrate that endoglin is an essential component of thereceptor complex in mediating the effects of TGF-β₁ and TGF-β₃, villousexplants were preincubated with either antisense or antibody to endoglinto trigger EVT differentiation. After an overnight incubation, exogenousTGF-β₁, TGF-β₂ or TGF-β₃ were added at a concentration of 10 ng/ml.Explants were metabolically labelled at day 5 of culture and FNsynthesis was measured. Phospholmager analysis demonstrated that bothantibody and antisense to endoglin significantly stimulated FNsynthesis. Addition of exogenous TGF-β₁ and TGF-β₃ to explant culturesincubated with antisense ON or antibody to endoglin, which binds bothisoforms, did not alter the stimulatory effect of antisensc ON andantibody to endoglin on FN synthesis. In contrast, addition of TGF-β₂,which does not interact with endoglin, overcame the antibody andantisense ON stimulatory effect on FN synthesis. TGF-β₂, but not −β₁ and−β₃, inhibited also the EVT outgrowth and migration induced by theantisense endoglin treatment.

[0129] Discussion

[0130] Treatment of human villous explants from 5-8 weeks gestation withantibodies and antisense oligonucleotides to endoglin stimulated EVTdifferentiation along the invasive pathway. This was manifested by 1) asignificant increase in EVT outgrowth and migration, 2) an increase infibronectin production 3) stem villous trophoblast proliferation and 4)a switch in integrin expression similar to that observed in vivo duringformation of anchoring villi. These data suggest that endoglin regulatesEVT differentiation during placental development. Endoglin, which isexpressed in vivo in the transition area here polarized trophoblastsbreak through the syncytium and begin forming columns of non-polarizedcells, appears to be a key molecule in mediating the inhibition oftrophoblast differentiation.

[0131] During the first trimester of gestation TGF-β is colocalized withone of its natural inhibitors, decorin, in the ECM of decidual tissue,suggesting that this proteoglycan may aid TGF-β storage or limit itsactivity within the decidual ECM (12). The findings described hereinsuggest that TGF-β produced by the villi is a negative regulator oftrophoblast differentiation along the invasive pathway. The expressionof endoglin at the transitional zone from polarized to non-polarizedtrophoblasts appears essential to the mediation of this negativeregulation. Blocking endoglin expression in this transition phasetriggers EVT outgrowth and migration and FN production. Thus,trophoblast invasion, characteristic of normal human placentation, isdependent on an intricate balance between positive and negativeregulators. The data herein indicate that endoglin is a criticalnegative regulator of this system. Therefore, inappropriate expressionor function of endoglin may contribute to the major complications ofpregnancy such as preeclampsia or chonocarcinoma, associated withabnormal trophoblast invasion and placenta development.

EXAMPLE 2

[0132] The present experiments were conducted to define the precisecomponents that endogenously regulate trophoblast invasion. Using humanvillous explants of 5-7 weeks gestation it was observed that whiletrophoblast cells remain viable they do not spontaneously invade intothe surrounding matrigel. In contrast, trophoblast cells from 9-13 weeksexplants spontaneously invade the matrigel in association with anupregulation of fibronectin synthesis and integrin switching.Trophoblast invasion at 5-7 weeks can be induced by incubation withantisense to TGF-β₃, TGFβ receptor I (ALK-1) or TGFβ receptor II. Onlyminimal invasion occurred in response to antisense to TGFβ₁ andantisense TGFβ₂ failed to induce invasion. These data suggest thatTGF-β₃ via the ALK-1-receptor II complex is a major regulator oftrophoblast invasion in vitro. To determine whether this system may alsooperate in vivo, immunohistochemical staining was conducted for TGF-β1and -3 and for TGFβ receptor I and II in trophoblast tissue from 5-13weeks of gestation. Strong positive immunoreactivity was observed forTGF-β₃ in both cyto- and syncytiotrophoblast from 5-9 weeks of gestationbut immunoreactivity was markedly reduced by 12-13 weeks. Expression ofTGFβ₁ was absent at 5 weeks, and transiently expressed at around 8 weeksof gestation. TGF receptor I and II immunoreactivity was strong between5-8 weeks but was not present at 12-13 weeks. Thus, the presence of TGFAand its receptors at 5-8 weeks at a time when there is no spontaneoustrophoblast invasion in vitro and the absence of these molecules at12-13 weeks when spontaneous in vitro invasion occurs is consistent witha major role for TGF-β₃ as an endogenous inhibitor of trophoblastinvasion.

EXAMPLE 3

[0133] Studies were carried out to determine if shallow trophoblastinvasion in preeclampsia was associated with an abnormally sustainedinhibition of invasion by TGF-β. In particular, theexpression/distribution of the different TGF-β isoforms and theirreceptors was investigated using immunohistochemical analysis in normalplacentae at 7-9 weeks (at the onset of trophoblast invasion) at 12-13weeks (the period of peak invasion), in control placentae between 29 and34 weeks and in preeclamptic placentae ranging from 27 to 34 weeks. Innormal placentae. TGF-β₃ expression was markedly reduced with advancinggestational age. Expression was high in cyto- and syncytiotrophoblastcells at 7-9 weeks of gestation but was absent in villous tissue at12-13 weeks and at 29-34 weeks of gestation. A similar decline inpositive immunoreactivity against TGF-β receptor I and II was alsoobserved over this time period. In contrast, in preeclamptic placentaebetween 27-34 weeks of gestation, strong staining for TGF-β₃ and itsreceptors was present in syncytiotrophoblast and stromal cells,immunopositive reactivity was not detected against TGF-β₁ or TGF-β₂ ineither normal or preeclamptic placentae. These data indicates thatpreeclampsia may result from a failure of trophoblast cells todownregulate expression of TGF-β₃ and its receptors which continue toexert an inhibitory influence on trophoblast invasion into the uterinewall.

EXAMPLE 4

[0134] Materials and Methods

[0135] RT-PCR and Southern Blot Analysis

[0136] Total RNA was extrace from the placenta, reverse transcribed andamplified by 15 cycles of PCR using TGFβ isoform specific primers.RT-PCR products were analysed by Southern blotting using ³²P-labelledTGFβ cDNAs. The primer set chosen for amplification of TGFβs were basedon human mRNA sequences. Primers used for amplification were: (a) TGFβ₁cDNA: (forward primer): 5′-GCCCTGGACACCAACTATTGCT-3′. (revesed primer):5′-AGGCTCCAAATGTAGGGGC AGG-3′ (predicted product size=161 bp); (b) TGFβ₂cDNA (forward primer): 5′-CATCTGGTCCCGGTGGCGCT-3′, (reversed primer):5′-GACGATTCTGAAGTAGGG-3′ (predicted product size=353 bp); (c) TGFβ₃cDNA: (forward primer): 5′-CAAAGGGCTCTGGTGGTCCTG-3′, (reversed primer):5′-CTTAGAGGTAATTCCCTTGGGG-3′ (predicted product size=374 bp): (c)β-actin cDNA: (forward primer): 5′-CTTCTACAATGAGCTGGGTG-3′. (reversedprimer): 5′-TCATGAGGTAGTCAGTCAGG-3′ (predicted product size=307 bp). Theidentity of the PCR reaction products was also confirmed by sequencing.

[0137] Immunohistochemistry

[0138] Placental tissue was processed for immunocytochemistry aspreviously described (I. Caniggia et al Endocrinology, 138, 3976 1997).Purified rabbit polyclonal antibody directed against TGFβ₁, TGFβ₂ andTGFβ₃ (Santa Cruz Biotechnology, Santa Cruz, Calif, were used at 1:50dilution. Sections (7 μm) were stained using the avidin-biotinimmunoperoxidase method (I. Caniggia et al Endocrinology, 138, 39761997). Control experiments included replacement of primary antibodieswith antiserum preincubated with an excess of TGFβs (competing peptide)or with blocking solution [5% (vol/vol) NGS and 1% (wt/vol) BSA].

[0139] Human Villus Explant Culture System

[0140] Villous explant cultures were established as described previously(I. Caniggia et al Endocrinology, 138, 3976 1997, O.Genbacev et al.Placenta 13:439, 1992) from first trimester human placentae (5-10 weeksgestation) or from preeclamptic and age-matched control placentae (30and 32 weeks of gestation) after collection according to ethicalguidelines. The preeclamptic group was selected according to bothclinical and pathological criteria (L. Chesley, Obstet. Gynecol, 65,423, 1985). Following an overnight period in serum-free DMEM/F12,explants were cultured in media containing antisense or senseoligonucleotides (10 μM) for up to 6 days (with changes ofmedia/oligonucleotides every 48 hours). Phosphorothioateoligonuclcotides of 16 base pairs targeted against sequences adjacent tothe AUG initiation codon of different human TGFβ isoforms mRNA weresynthesized as follows: TGFβ₁ 5′-CCCCGAGGGCGGCATG-3′ and5′-CATGCCGCCCTCGGGG-3′, respectively; TGFβ₂ 5′-CACACAGTAGTGCATG-3′ and5′-CATGCACTACTGTGTG-3′; TGFβ₃ 5′-CCTTTGCAAGTGCATC-3′ and5′-GATGCACTTGCAAAGG-3′.

[0141] Fibronectin Synthesis

[0142] To measure fibronectin synthesis on day 5 explants were culturedin the presence of 25 μCi/ml of [³⁵S]methionine/cysteine for 18 hours.Conditioned culture media were collected and diluted with an equalamount of 25 mM Tris-HCl buffer, pH 7.4. 0.15 M NaCl and 0.5% (v/v)Triton X-100 and fibronectin was isolated using gelatin-Sepharose aspreviously described (I. Caniggia et al Endocrinology, 138, 3976 1997,E. Engvall et al Int. J. Cancer, 20: 1, 1977). Radiolabeled fibronectinwas revealed by autoradiography and quanutated using a Phospholmager(410A and Image Quant software, Molecular Dynamics).

[0143] Gelatinolytic Activity

[0144] Analysis of gelatinolytic activity was performed using 10%polyacrylamide gel (wt/vol) impregnated with 0.1% gelatin (NOVEX, SanDiego, Calif.) as previously described (I. Caniggia et al Endocrinology,138, 3976 1997). For Western blot analysis of metalloproteasesexpression, 5 μl of conditioned media were subjected to gelelectrophoresis using 10% polyacrylamide gels. Proteins were thenblotted to Westran PVDF membrane. Primary antibodies were used at 1:100dilution, and detected using horse radish peroxidase conjugatedantimouse IgG (Amerham 1:10,000 fold dilution) and enhanced bychemiluminescence (ECL, Amerham).

[0145] Results

[0146] The expression of TGFβ around 9-12 weeks of pregnancy and itsrelationship to trophoblast invasion and subsequently preeclampsia wereinvestigated. Using low cycle RT-PCR followed by Southern blot analysisall three isoforms of TGFβ were found to be expressed during the firsttrimester (FIG. 3A). However, while transcripts corresponding to TGFβ₁and TGFβ₂ were uniformly expressed throughout this period, theexpression of TGFβ₃ exhibited a striking pattern of developmental ortemporal regulation. TGFβ₃ mRNA levels were relatively low at 5-6 weeks,increased markedly between 7 and 8 weeks, and then fell precipitously at9 weeks. This pattern of expression for the TGFβ₃ isoform was confirmedat the protein level by immunohistochemistry (FIG. 3B). TGFβ₃ waslocalized to cyto and syncytiotrophoblasts within the villous and alsoto cytotrophoblasts within the invading column (FIG. 3B). TGFβ₃ wasnoticeably absent in those cytotrophoblast cells at the transitionbetween polarized and nonnpolarized cells at the proximal site of theforming column. Importantly, the down-regulation of TGFβ₃ around 9 weeksis temporally associated with the period of maximal trophoblast invasionin vivo and the expression of markers of cytotrophoblast invasion,including switching of integrin isoforms (Damsky, C. H. et alDevelopment 120:3657, 1994), synthesis of matrix ligands for theseintegrins (P. Bischof, L. Haenggeli A. Campana, Human Reprod. 10, 734.(1995), M. J. Kupferminc, A. M. Peaceman, T. R. Wigton, K. A. Rehnberg,M. A. Socol, Am.J. Obstet. Gynecol. 172, 649 (1995)) and upregulation ofgelatinase A (MMP2) and gelatinase B (MMP9) activity (C. I. Librach, etal. J.Biol. Chem. 269, 17125. (1994)).

[0147] To determine the functional significance of the TGFβ expressionpatterns, a human villus explant culture system was used which mimicsclosely the normal pattern of trophoblast invasion in vivo (I. Caniggia,C. V. Taylor, J. W. K. Ritchie. S. J. Lye, M. Letarte, Endocrinology,138, 4977 (1997), O. Genbacev, S. A. Schubach, R. K. Miller, Placenta13, 439. (1992)). Morphologic (EVT outgrowth) and biochemical(fibronecun [FN] synthesis and gelatinase activity) indices oftrophoblast invasion were monitored in response to antisense (AS)induced suppression of TGFβ isoform expression in explants at 5-8 weeksof gestation. Explants exposed to AS TGFβ₃ (but not TGFβ₁ or TGFβ₂)displayed prominent EVT outgrowth from the distal end of the villous tip(FIG. 4A). This morphologic response was associated with a significantincrease in FN synthesis (FIG. 4B. and FIG. 4E) and gelatinase activity(FIG. 4D). The specificity of the AS TGFβ₃ response was demonstrated byreversal of both morphologic and biochemical indices when AS-treateexplants were cultured in the presence of TGFβ₃ but not TGFβ₁ (FIG. 4C).The induction of FN synthesis by AS TGFβ₃ at 5-8 weeks was lost at 9-13weeks (FIG. 4E) further demonstrating the specificity of the AS actionas TGFβ₃ is not expressed in villous trophoblast at 9-12 weeks.

[0148] These functional data together with the temporal-spatualexpression patterns strongly suggest that down-regulation of TGFβ₃around 9-12 weeks is required for optimal trophoblast invasion indicatethat a failure to down-regulate TGFβ₃ expression is the basis of limitedtrophoblast invasion found in preeclampsia. Significantly higher levelsof mRNA encoding TGFβ₃ (but not TGFβ₁ or TGFβ₂) were found inpreeclamptic versus control placentae (FIG. 5A). Immunoreactive TGFβ₃intensively labelled syncytio and cytotrophoblasts in villous tissuesfrom preeclamptic patients while little or no immunoreactivity waspresent in the age-matched controls (FIG. 5B). Elevated levels of FNmRNA and a failure to complete integrin switching (i.e the trophoblastremain positive for α₅ and fail to express α₁ were also obverved inpreeclamptic placentae. These data suggest that the trophoblasts frompreeclamptic placenta are arrested at a relatively immature phenotypepossibly due to a failure to undergo complete differentiation along theinvasive pathway during the first trimester of gestation.

[0149] To determine whether there was functional significance associatedwith overexpression of TGFβ₃ in preeclamptic placentae, the pattern oftrophoblast differentiation along the invasive pathway in explants fromcontrol and preeclamptic patients was analyzed. When cultured onmatrigel, explants from non-preeclamptic patients showed formation ofEVT columns which spontaneously invaded into the surrounding matrigel.In contrast, explants from preeclamptic placentae failed to exhibit EVToutgrowth or invasion (FIG. 6A). These data are consistent with the viewthat preeclampsia is associated with reduced invasive capability oftrophoblasts. Of critical importance to the investigation was whetherthis reduced invasive capability was due to the overexpression of TGFβ₃.Therefore the differentiation of villous explants from preeclampticpatients cultured in the presence of AS TGFβ₃ was monitored. In contrastto untreated or sense-treated controls, treatment of explants frompreeclamptic patients with AS TGFβ₃ restored the invasive capability, asdemonstrated by the formation of EVT columns migrating through thematrigel (FIG. 6A). The invasive nature of this phenotype was confirmedby the finding that explants treated with AS TGFβ₃ acquired theexpression of gelatinase B/MMP9, an enzyme which is normally onlyexpressed in trophoblast cells that are highly invasive (FIG. 6B andFIG. 6C).

[0150] The data presented here demonstrate not only that abnormalitiesin TGFβ₃ expression ame associated with preeclampsia but also thatdown-regulation of TGFβ₃ with antisense oligonucleotides restores theinvasive capability of preeclamptic trophoblasts. The data areconsistent with a model of normal placentation in which down-regulationof TGFβ₃ expression in trophoblast around 9 weeks of pregnancy permitsdifferentiation of trophoblast to EVT that form the anchoring villi andfrom which derive the α1-integrin positive EVT which invade deep intothe maternal uterus. This invasion contributes to the remodelling of theuterine spiral aeries and ultimately enables the establishment ofincreased vascular perfusion of the placenta. In placentae predisposedto preeclampsia, TGFβ₃ expression remains abnormally elevated andtrophoblasts remain in a relatively immature state of differentiation.As a direct consequence, trophoblast invasion into the uterus is limitedand uteroplacental perfusion is reduced. This conclusion is consistentwith the clinical manifestations of preeclampsia, including shallowtrophoblast invasion into the uterus and abnormally high uteroplacentalvascular resistance.

EXAMPLE 5

[0151] Role of O₂ Tension in Trophoblast Invasion

[0152] The role of oxygen tension in regulating trophoblastdifferentiation along the invasive pathway has been investigated. Thedata indicate that expression of hypoxia inducible factor HIF-1αparallels that of TGFβ₃ in first trimester trophoblast (i.e. peaks at6-8 weeks but decreases after 9-10 weeks when oxygen tension increases(FIG. 7A). The presence of putative HIF-1 binding sites in the promoterregion of the TGFβ₃ gene suggests that inducuon of HIF-1α by low PO₂(around 6-8 weeks) up regulates TGFβ₃ tanscription and blocks atrophoblast invasion. A failure of the system to down-regulate at 9-12weeks (either due to a block in response to normoxia or the absence ofan increase in oxygen tension) could lead to shallow invasion andpredispose to preeclampsia. This is supported by data showing thatexpression of HIF-1α is dramatically increased in placentas ofpreeclamptic patients when compared to age-matched control tissue (FIG.7B). In FIG. 7A and 7B mRNA HIF-1α expression was assessed by using lowcycle RT-PCR followed by Southern blot analysis. This is also supportedby FIG. 8 showing the effect of low oxygen tension of TGFβ₃ and HIF-1αexpression in villous explants; FIG. 9 showing the effect of low oxygentension on villous explant morphology; and FIG. 10 showing the effect ofantisense to HIF-1α on villous explant morphology.

EXAMPLE 6

[0153] TGFβ₃ Signals Through a Receptor Complex

[0154] In addition to endoglin, evidence indicates that TGFβ₃ signalsthrough a receptor complex which includes RI (ALK-1) and RII. While TGFβR-I (ALK-5) and TGFβ R-II are expressed throughout the villi and dcciduaat 9-10 weeks gestation: they are absent from the base of the proximalcolumns of the anchoring villi at the transition zone between thevillous and the invading EVT, exactly at the site where endoglin isupregulated. This dramatic change in TGF-β receptor expression suggeststhat EVTs within the columns in situ are not subject to the inhibitoryactions of TGFβ but via R-I and R-II they do come under the control ofthis ligand upon entering the decidua. The potential clinical importanceof the TGFβ receptor system in trophoblast invasion is highlighted bydata demonstrating that beside TGFβ₃, R-I is expressed at greater levelsin trophoblast tissue of preeclamptic patients when compared to that inage-matched control placenta. Antisense disruption of R-I (ALK-1) andR-II expression stimulated trophoblast outgrowth/migration and FNsynthesis. In contrast, antisense to R-I (ALK-5) inhibited FN synthesis.

[0155] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0156] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

[0157] Below full citations are set out for the references referred toin the specification and detailed legends for the figures are provided.

[0158] Full Citations for References Referred to in the Specification

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[0160] 2. Zhou Y, Damsky C H, Chiu K, Roberts J M, Fisher S J. 1993.Preeclampsia is associated with abnormal expression of adhesionmolecules by invasive cytotrophoblasts. J. Clin. Invest. 91: 950-960.

[0161] 3. Aplin J D. 1991. Implantation, trophoblast differentiation andhaemochorial placentation: mechanistic evidence in vivo and in vitro. J.Cell Science. 99: 681-692.

[0162] 4. Damsky C H, Fitzgerald M L, Fisher S J. 1992. Distributionpatterns of cxtracellular matrix components and adhesion receptors areintricately modulated during first trimester cytotrophoblastdifferentiation along the invasive pathway, in vivo. J. Clin. Invest.89: 210-222.

[0163] 5. Bischof P, Redard M, Gindre P, Vassilakos P, Campana A. 1993.Localization of alpha2, alpha5 and alpha6 integrin subunits in humanendometrium, decidua and trophoblast. Eur. J. of Obst. and Gyn. andRepr. Biol. 51: 217-226.

[0164] 6. St-Jacques S, Forte M, Lye S J, Letarte M. 1994. Localizationof endoglin, a transforming growth-factor-b binding protein, and of CD44and integrins in placenta during the first trimester of pregnancy, BiolReprod. 51: 405-413.

[0165] 7. Fisher S J, Cui T. Zhang L et al. 1989. Adhesive anddegradative properties of human placental cytotrophoblast cell in vitro.J. Cell. Biol. 109: 891-902.

[0166] 8. Librach C L, Feigenbaun S L, Bass K E, et al. 1994.Interleukin-1b regulates human cytotrophoblast metalloproteinaseactivity and invasion in vitro. J. Biol. Chem. 269: 17125-17131.

[0167] 9. Bass K E, Morrish D, Roth I, et al. 1994 Human cytotrophoblastinvasion is unregulated by epidermal growth factor: evidence thatparacrine factors modify this process. Devel. Biol. 164: 550-561.

[0168] 10. Graham C H, Lysiak J J, McCrae K R, Lala P K. 1992.Localization of transforming growth factor-b at the human fetal-maternalinterface: role in trophoblast growth and differentiation, Biol. Reprod.46: 561-572.

[0169] 11. Graham C H, Lala P K. 1991. Mechanism of control oftrophoblast invasion in situ. J. Cell. Physiol. 148: 228-234.

[0170] 12. Lysiak J J, Hunt J, Pringle J A, Lala P K. 1995. Localizationof transforming growth factor b and its natural inhibitor decorin in thehuman placenta and decidua throughout gestation. Placenta, 16: 221-231.

[0171] 13. Irving J A, and Lala P K, 1995. Functional role of cellsurface integrins on human trophoblast cell migration: regulation byTGF-b, IGF-II, and IGBP-I. Exp. Cell. Res. 217: 419-427.

[0172] 14. Cheifetz S, Bellon T, Cales C, et al. 1992. Endoglin is acomponent of the transforming growth factor-b receptor system in humanendothelial cells. J. Biol. Chem. 267: 19027-19030.

[0173] 15. Wrana J L, Attisano L, Wieser R, Ventura F, Massague J. 1994.Mechanisms of activation of the TGF-b receptor. Nature. 370: 341-347.

[0174] 16. Mitchell E J, Fitz-Gibbon L, O'Connor-McCourt M D. 1992.Subtypes of betaglycan and type I and type II transforming growthfactor-b (TGF-b) receptors with different affinities for TGF-b1 andTGF-b2 are exhibited by human placenta trophoblasts. J. Cell. Physiol.150: 334-343.

[0175] 17. Gougos A, St-Jacques S, Greaves A, et al. 1992.Identification of distinct epitopes of endoglin, an RGD-containingglycoprotein of endothelial cells, leukemic cells, andsyncytiotrophoblasts. Int. Immunol. 4: 83-92.

[0176] 18. Yamashita H, Ichijo H, Grimsby S, Moren A, ten Dijke P, andMiyazono K. 1994. Endoglin forms an heteromeric complex with thesignalling receptors for transforming growth factor-b. J. Biol. Chem.269: 1995-2001.

[0177] 19. Zhang H, Shaw A R E, Mak A, and Letarte M. 1996. Endoglin isa component of the TGF-b receptor complex of human pre-B leukemic cells.J. Immunol. 156: 565-573.

[0178] 20. Lastres P, Letamendia A, Zhang H, et al. 1996. Endoglinmodulates cellular responses to TGF-b 1. J. Cell Biol. 133: 1109-1121.

[0179] 21. Genbacev O, Schubach S A, Miller R K. (1992). Villous cultureof first trimester human placenta—Model to study extravilloustrophoblast (EVT) differentiation. Placenta. 13: 439-461.

[0180] 22. Quackenbush E J and Letarte M. 1985. Identification ofseveral cell surface proteins of non-T, non-B acute lymphoblasticleukemia by using monoclonal antibodies. J. Immunol. 134: 1276-1285.

[0181] 23. Gougos A, and Letarte M. 1990. Primary structure of endoglin,an RGD-containing glycoprotein of human endothelial cells, J. Biol.Chem. 265: 8361-8364.

[0182] 24. Malcolm A D B. 1992. Uses and applications of antisenseoligonucleotides: uses of antisense nucleic acids—an introduction.Bioch. Soc. Trans. 20: 745-746.

[0183] 25. Engvall E, and Ruoslahti E. 1977. Binding of soluble form offibroblast surface protein, fibronectin, to collagen. Int. J. Cancer.20: 1-5.

[0184] 26. Soubiran P, Hsi B-L, Lipinski M, Yeh C-J G, Vaigot P,Masseyeff R. 1986. Distribution of Trop 3 and 4 antigens as defined bymonoclonal antibodies raised against a human choriocarcinoma cell line.A.J.R.I.M. 12: 118-123.

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[0190] Detailed Figure Legends

[0191]FIG. 3 Expression of TGF-β isoforms in human placenta in the firsttrimester of gestation. (FIG. 3A) Message expression of TGFβ isoformswas assessed by low cycle RT-PCR followed by Southern blot analysisusing specific probes for TGFβ₁, TGFβ₂ and TGFβ₃ and the controlhouse-keeping gene β-actin. Note that TGFβ₃ expression increases around7-8 weeks gestation and declines thereafter. (FIG. 3B) Immunoperoxidasestaining of TGFβ₃ was performed in placental sections at 5, 8 and 12weeks of gestation. Sections of placental tissue of 5 weeks gestationshow positive immunoreactivity visualized by brown staining in thecytotrophoblast, syncytiotrophoblast and stromal cells of the chorionicvilli. Sections of placental tissue of 8 weeks gestation show strongpositive immunoraactivity in the cytotrophoblast, syncytiotrophoblast,and stromal cells. Note that TGF-B₃ was expressed in the non-polarizedtrophoblast within the column (EVT, thin arrow) but was absent in thetransitional zone where polarized cells become unpolarized (thickarrows). Sections of placenta at 12 weeks gestation demonstrate low orabsent TGF-β₃ immunoreactivity in the villi. There is noimmunoreactivity when antiserum was preincubated with an excess of TGFβ₃competing peptide (8 weeks, control).

[0192]FIG. 4 Antisense TGFβ₃ stimulates trophoblast migration,fibronectin production and gelatinase activity. Explants of 5-8 weeksgestation were treated for 5 days with 10 μM antisense oligonucleotidesto TGFβ₃ (AS-β3). AS-β₃ plus 10 ng/ml recombinant TGFβ3 (AS-β3+β3) orAS-β3 plus recombinant TGFβ1 (AS-β3+β1). Control experiments were run inparallel using sense TGF-β₃ (S-β3) or medium alone (FIG. 4C). (FIG. 4A)Shown is a representative experiment demonstrating that addition ofrecombinant TGFβ₃ to antisense TGFβ₃ treated explants (AS-β3+β3)abolishes the antisense stimulatory effect on trophoblasts budding andoutgrowth (arrows). (FIG. 4B) Similar reversal effect on AS-β3stimulatory effect by exogenous TGFβ₃ was demonstrated also forfibronectin synthesis. Representative analysis of triplicate samplesfrom a single experiments is shown. The position of the marker withM_(r)=200×10³ is indicated. Lanes 1-3, S-β3 treated explants; lanes 4-6,AS-β3 treated explants; lanes 7-9, AS-β3+β3 treated explants. (FIG. 4C)Changes in fibronectin estimated after normalization to controlcultures. Antisense TGFβ₃ treatment (AS-β3, solid bar) significantlyincreased (p<0.05; one-way ANOVA followed by Student-Newman-Keuls testfor non-paired groups) the amount of labelled fibronectin compared toboth medium alone (FIG. 4C, open bar) or sense (S-β3, cross bar).Addition of exogenous TGFβ₃ (AS-β3+β3 squares bar) but not TGFβ₁(AS-β3+β1 cross hatched bar) to the antisense treated explants abolishedthe antisense stimulatory effect on fibronectin production,demonstrating the specificity of the action of TGFβ₃. (FIG. 4D)Gelatinase activity in conditioned media of explants teated with senseor antisense oligonucleotides to TGFβ3. Arrows indicate positions ofgelatinases activity (MMP2: 60, 68; MMP9: 84 and 92, kDa). (FIG. 4E) Theantisense TGFβ₃ stimulatory effect on fibronectin production is lostafter 9 weeks of gestation. Explants of 6 and 10 weeks gestation weretreated with 10 μM antisense (AS-β3) or control sense (S-β3)oligonucleotides to TGFβ₃. Newly synthesized fibronectin was isolatedfrom the medium as dessibed above. Representative analysis of triplicatesamples from a single experiment is shown. Lanes 1-3 and 7-9, S-β3treated explants; lanes 4-6 and 10-12, AS-β3 treated explants.

[0193]FIG. 5 TGFβ₃ is overexpressed in preeclamptic placentae. (FIG. 5A)Message expression of TGFβ isoforms, α₅ integrin receptor andfibronectin in preeclamptic (PE) and age-matched control placentae (FIG.5C) was assessed by low cycle RT-PCR followed by Southern blot analysisusing specific probes for TGFβ₁, TGFβ₂, TGFβ₃, α₅, fibronectin and thecontrol house-keeping gene β-actin. Note that TGFβ₃, α₅ and fibronectin,but not TGFβ₁ or TGFβ₂, expression were higher in preeclamptic placentaewhen compared to age-matched control. (FIG. 5B) Immunoperoxidasestaining of TGFβ₃ was performed in placental sections from normalpregnancies and pregnancies complicated by preclampsia. Sections ofnormal placental tissue of 29, 31 and 33 weeks of gestation showlow/absent TGFβ₃ immunoreactivity in cells of the chorionic villi.Sections of preeclamptic placental tissue of the same gestation showstrong positive immunoreactivity visualized by brown staining in thecytotrophoblast, syncytiotrophoblast and stromal cells of the chorionicvilli. Control experiments were performed using antiserum preabsorbedwith an excess of peptide.

[0194]FIG. 6A Antisense oligonucleotides to TGFβ₃ induces the formationof columns of trophoblast cells in prceclamptic villous explants.Villous explant cultures were prepared from preeclanptic and age-matchedcontrol placentae. Explants were mantained in culture in the presence ofeither control sense or antisense oligonucleotides to TGFβ₃ for 5 days.Morphological integrity was recorded daily. Explants from normalplacenta (32 weeks), exposed to sense oligonucleotides (S-β3)spontaneously form columns of trophoblast cells which migrate and invadeinto the surrounding Matrigel (arrows), while explants from preeclampticplacenta (32 weeks) exposed to sense oligonucleotides do not. Incontrast, antisense treatment (AS-β3) triggers the formation of invadingtrophoblast columns (arrows) in preeclamptic placentae.

[0195]FIG. 6B and FIG. 6C. Antisense oligonucleotides to TGFβ₃ triggersgelatinasc activity and expression in preeclamptic villous explants.Explants of 32 weeks gestation from preeclamptic placentae were treatedwith antisense (AS-β3) or control sense (S-β3) oligonucleotides to TGFβ₃for 5 days. Samples of conditioned medium were collected at day 5 andsubjected to analysis by gelatin Zymography (FIG. 6B) or Westernblotting with MMP9 antisera (FIG. 6C). Arrows indicate positions ofgelatinases activity (MMP-2: 60, 68; MMP-9: 84 and 92, kDa).

We claim:
 1. A method for detecting, preventing, and/or treating acondition requiring regulation of trophoblast invasion comprisingmodulating TGFβ3, receptors of cytokines of the TGFβ family, HIF-1α, oroxygen tension.
 2. A method for diagnosing in a subject a conditionrequiring regulation of trophoblast invasion comprising detectingTGF-β3, receptors of cytokines of the TGFβ family, or HIF-1α in a samplefrom the subject.
 3. A method for diagnosing increased risk ofpreeclampsia in a subject comprising detecting TGF-β₃ in a sample fromthe subject.
 4. A method as claimed in claim 3 which comprises (a)collecting a sample from the subject: (b) measuring the levels of TGF-β₃in the sample; and (c) comparing the levels or TGF-β₃ in the sample tothe levels in women with normal pregnancies.
 5. A method as claimed inclaim 4 wherein the levels of TGF-β₃ are measured in a sample from thesubject during the first trimester of pregnancy.
 6. A method ofregulating trophoblast invasion comprising inhibiting or stimulatingTGF-β₃, receptors of cytokines of the TGFβ family, HIF-1α, or oxygentension.
 7. A method for increasing trophoblast invasion in a subjectcomprising administering an effective amount of an inhibitor of (a)TGF-β₃, (b) receptors of cytokines of the TGFβ family, or (c) HIF-1α. 8.A method as claimed in claim 7 wherein the inhibitor is antisense toTGFβ₃ or antisense to HIF-1α.
 9. A method as claimed in claim 7 whereinthe inhibitor is an antibody to TGFβ₃.
 10. A method as claimed in claim7 wherein the inhibitor is decorin, fetuin, α₂-macroglobuliln, orthyroglobulin, or peptides derived from sites on the compounds that bindto TGFβ3.
 11. A method for reducing trophoblast invasion in a subjectcomprising administering an effective amount of (a) TGF-β₃, (b)receptors of cytokines of the TGFβ family, (c) HIF-1α, or (d) astimulator of (a), (b), or (c).
 12. A method for treating a womansuffering from, or who may be susceptible to preeclampsia comprisingadministering therapeutically effective dosages of an inhibitor of (a)TGF-β₃, (b) receptors of cytokines of the TGFβ family, or (c) HIF-1α.13. A method for monitoring or treating choriocarcinoma in a subjectcomprising administering herapeutically effective dosages of (a) TGFβ₃,(b) a receptor of cytokines of the TGFβ family, (c) HIF-1α and/or (d)stimulators of (a), (b) or (c).
 14. A method for evaluating a compoundfor its ability to regulate trophoblast invasion comprising the stepsof: (a) reacting TGFβ₃ and a receptor of a cytokine of the TGFβ family,and a test substance, wherein the TGFβ₃ and receptor of a cytokine ofthe TGFβ family, are selected so that they bind to form aligand-receptor complex; and (b) comparing to a control in the absenceof the substance to determine if the substance stimulates or inhibitsthe binding of TGFβ₃ to the receptor and thereby regulates trophoblastinvasion.
 15. A method for evaluating a substance for its ability toregulate trophoblast invasion comprising the steps of: (a) reactingTGFβ₃, HIF-1α, and a test substance, wherein the TGFβ₃ and HIF-1α bindto form a TGFβ₃-HIF-1α complex; and (b) comparing to a control in theabsence of the substance to determine if the substance stimulates orinhibits the binding of TGFβ₃ to HIF-1α and thereby regulatestrophoblast invasion.
 16. A receptor complex comprising TGFβ R-I(ALK-1)-TGFβ RII-endoglin.
 17. A composition for regulating trophoblastinvasion comprising an inhibitor of (a) TGF-β₃, (b) receptors ofcytokines of the TGFβ family, or (c) HIF-1α in an amount effective toreduce trophoblast invasion, and a carrier, diluent or excipient.
 18. Acomposition as claimed in claim 17 wherein the inhibitor is antisense toTGFβ₃ or antisense to HIF-1α.
 19. A composition as claimed in claim 17wherein the inhibitor is an antibody to TGFβ₃.